The epithelium is the membranous cellular tissue that covers the surface or lines a tube or cavity of an animal body. The epithelium serves to enclose and protect the other parts of the body and may produce secretions and excretions and may be associated with assimilation as seen in the gastrointestinal tract. The epithelium is one of the four primary tissues of the body, which constitutes the epidermis and the lining of respiratory, digestive and genitourinary passages. The cornea, which is the transparent part of the coat of the eyeball that covers the iris and pupil and admits light to the interior, is also a tissue that is made of epithelial cells.
The functions of epithelia are varied and include: (1) protective function, by completely covering the external surface (including the gastrointestinal surface, the surface of the whole pulmonary tree including the alveoli and the eye); (2) secretory function, by secreting fluids and chemical substances necessary for digestion, lubrication, protection, excretion of waste products, reproduction and the regulation of metabolic processes of the body; (3) absorptive function, by absorbing nutritive substances and preserving water and salts of the body; (4) sensory function, by constituting important parts of sense organs, especially of smell and taste; and (5) lubricating function, by lining all of the internal cavities of the body, including the peritoneum, pleura, pericardium and the tuncia vaginalis of the testis.
The growth of human epithelial cells without the use of companion-cells, protein growth factors, feeder layers, serum components or organotypic substrates is the advancement in the state of the art that is this invention. Traditionally, tissue culture of normal epithelial cells has been attempted in a variety of commercially available media designed for the growth of less fastidious types of cells, i.e., malignant cells transformed in vitro from cell lines derived from human or non-human tissues, cell lines developed from human or non-human tumors, or cell lines developed for human or non-human embryonic mesenchymal cell types. In contrast, the culture of normal human epithelial stem cells has presented many difficulties not the least of which is the inexorable tendency for these cells to undergo uncontrolled, irreversible, terminal differentiation with the consequent loss of cell division capacity.
A significant development made by Tsao et al. is the formulation of a nutrient medium supplemented with specified growth factors and hormones allowed for the growth of human epidermal cells. See Tsao, M. C. et al., J Cellular Physiol. 110:219-229 (1982). The Tsao medium has been designated MCDB 152. Further refinements of this medium lead to the development of a medium known as MCDB 153. See Boyce, S. T. and Ham, R. G., J Invest. Dermatol. 81:33-40 (1983). The use of these media permitted a more accurate characterization of the necessary growth factors, hormones and Ca2+ requirements for retention of high cloning efficiency which is necessary to maintain proper genetic programming for continued subculture of pluripotent basal epidermal stem cells. See Wille, J. J. et al., J Cellular Physiol. 121:31-44 (1984).
The use of serum in cell culture medium provides a complex mixture of growth factors and differentiation-inducing factors. See Pittelkow, M. R. et al., J Invest, Dermatol. 86:410-417 (1986). Pittelkow et al. reported that serum, known to contain fibroblastic cell growth factors, e.g., platelet-derived growth factor, was an inhibitor of basal epidermal cell growth. Further, the differentiation-inducing factors in serum could be equated with serum's content of β-transforming growth factor, (β-TGF). See Shipley, S. D. et al., Cancer Res. 46:2068-2071 (1986). It has also been reported that normal human keratinocytes actually produce their own growth factors. That is, proliferating basal cells are stimulated to secrete α-transforming growth factor (α-TGF) in the presence of added epidermal growth factor (EGF) and decrease production of α-TGF at high cell densities near confluence. Under the latter condition, the arrested cells secrete an inactive form of β-TGF. See Coffey, R. J. et al., Nature 328:817-820 (1987). These considerations led the inventor to the idea that the natural mechanism of growth stimulation and its regulation in cultured epithelia cells could be accomplished through manipulation of the various media components and that such manipulation would also eliminate the need for an organic substrate or organotypic matrix as well.
Judd et al. discuss a keratinocyte growth medium designated keratinocytes-SFM in an article entitled: “Culture of Human Keratinocytes in Defined Serum Free Medium”, Focus, 19, No. 1, Pgs. 1-5. This serum-free media is also disclosed in a Gibco Product brochure. However, the actual composition of the SFM media is not disclosed other than it does not contain the growth promoting additives insulin, epidermal growth factor and fibroblast growth factor.
An article by Wille et al., in J Dental Research, 68:1019 (1989) entitled “Serum Free Cultures of Normal Human Gingival Keratinocytes (HGK)” discusses the successful in vitro culturing of human gingival keratinocytes in MCDB 153 medium, supplemented with 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, 0.5 mM hydrocortisone, 5 μg per ml epidermal growth factor, 5 μg per ml insulin and 35 μg per ml bovine pituitary extract protein where the presence of these proteins is necessary, but their function is unknown in this heterogeneous tissue extract mixture. Wille et al. in The Journal of Cellular Physiology, 150:52-58 (1992) in an article entitled “Effects of Growth Factors, Hormones, Bacterial Lipopolysaccharides and Lipotechoic Acids on the Clonal Growth of Urethreal Epithelial Cells in Serum Free Culture”, discloses the use of F-12 media containing bovine pituitary extract and bovine serum albumin for culturing cells isolated from human ureters, again where such tissue products have necessary but unknown effective components.
Chopra et al. in the Journal of Cellular Physiology, 130:173-181 (1987) entitled: “Propagation of Differentiating Normal Human Tracheobronchial Epithelial in Serum Free Medium” discloses the use of a medium similar to MCDB 151 except that it contains 5.4 μg per ml HEPES, 6.1 μg per ml sodium chloride, 0.3 μg per ml sodium acetate and 1 μg per ml sodium bicarbonate. These changes lowered the final osmolarity of the disclosed medium to 290 mosmols. The concentration of HEPES in the Chopra et al. solution was 28 mM.
U.S. Pat. No. 5,328,844 to Moore discloses a culture medium useful for establishing, growing and maintaining mammalian cells in culture, in particular for the establishment of culture of human, normal and malignant cells. The claimed media contains 4,500 mg per liter of HEPES and 5 mg per liter of insulin. This patent does not relate to nor disclose media useful for growth of normal epithelial cells.
In an article by Boisseau et al. entitled “Production of Epidermal Sheets in a Serum Free Culture Medium: A Further Appraisal of the Role of Extracellular Calcium”, Journal of Dermatological Science, 3 (1992), 111-120, the author discloses the serum-free media (MCDB 153) to grow keratinocyte monolayers in clonogenic conditions. The effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes was investigated.
U.S. Pat. No. 4,673,649 to Boyce et al. discloses a basal medium which was MCDB 152 supplemented with epidermal growth factor, transferin, insulin, hydrocortisone, ethanolamine, phosphoethanolamine and progesterone to obtain a medium for growth of human keratinocytes. The inventor of the present application in U.S. Pat. No. 5,292,655 demonstrates that progesterone inhibits optimal growth.
Wilke et al. in “Biologic Mechanisms for the Regulation of Normal Human Keratinocyte Proliferation and Differentiation”, American Journal of Pathology, Vol. 131, No. 1, April, 1988, describe a serum-free medium with low calcium concentrations on the order of 0.1 mM. These studies of Wilke et al. actually used MCDB 153 medium supplemented with insulin, EGF and protein of bovine pituitary extract where any effective components are unknown in the extract.
U.S. Pat. No. 5,232,848 to Wolfe et al. discloses a nutrient medium for both high and low density culture of a wide variety of non-epithelial cell lines and cell types. This patent discloses and claims a zwitterionic buffer such as HEPES at a concentration of 2.5×10−2 moles.
Boyce et al. in U.S. Pat. No. 4,940,666 discloses and claims a growth medium which is free of transferin, comprising complete MCDB 153, EGF and insulin.
Nissley et al. in “Growth and Differentiation of Cells in a Defined Environment”, pgs. 337-344 discloses that cells of embryonic and fetal origin produce IGF-1 and IGF-2 which may be important for the control of embryonic and fetal growth. The authors also suggest that the use of these cells could potentially stimulate the growth of the same or neighboring cells and thereby avoid the inclusion of such growth factors in a culture medium.
Boyce et al. in “Calcium Regulated Differentiation of Normal Human Epidermal Keratinocytes in Chemically Defined Clonal Culture and Serum Free Serial Culture”, in The Journal of Investigative Dermatology, 81:33S-40S (1983) discloses MCDB 153 supplemented with a number of growth factors and an optimum level of calcium at 0.3 mM for colony forming efficiency and a high calcium concentration of 1.0 mM for induction of stratification and terminal differentiation.
U.S. Pat. No. 5,326,699 to Torishima et al. discloses a serum-free medium for culturing animal epithelial cells comprising 8-14 μg per ml (53.6 mM -93.8 mM) of methionine, up to 0.1 mM of calcium in the form of calcium chloride and other conventional ingredients such as glucose, growth factors, buffers and the like.
U.S. Pat. No. 5,686,307 and U.S. Pat. No. 5,834,312 to Wille disclose a serum-free medium for culturing animal epithelial cells comprising the amino acid histidine (3.0-33 mg/L), isoleucine (3.0-33 mg/L), methionine (4.5-45 mg/L), tryptophan (4.0-44 mg/L) and tryosine (5.0-55 mg/L), NaCl (90-140 mM) and Hepes (14-22 mM) that is useful for the production of a living human skin and animal epithelia. This medium (HECK 109) requires EGF and IGF-1 as the only two protein growth for serial cultivation of proliferating cultures of normal human keratinocytes.
Varani et al. have reported that all-trans retinoic acid stimulates the growth of adult human keratinocytes cultured in a growth factor-deficient medium. Early passage keratinocytes were incubated for 1 or 2 days in a serum-free keratinocyte growth medium (MCDB 153) supplemented with EGF, insulin and BPE and 1.4 mM Ca2+ or in growth factor-deprived keratinocyte basal MCDB 153 medium. The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 ng/ml). Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in the growth-factor supplemented medium. By contrast, all-trans retinoic acid treatment of keratinocytes in growth-factor deficient medium, in which the cells were growth arrested, stimulated growth. Stimulation was observed in a serum-free medium lacking not only protein growth factors, but hydrocortisone, ethanolamine, and phosphoethanolamine. The rate of keratinocyte proliferation in the retinoid-stimulated cultures was approximately 35% of the maximal proliferation rate observed in growth factor supplemented medium. It should be noted that the optimal concentration of all-trans retinoic acid required to produce these effects was 0.5 ng/ml (1.6×10−6M). This is about 100-fold greater than the physiological concentration, and is present in amounts known to be damaging to cell membranes. Lower concentration of all-trans retinoic acid were ineffective.
In addition, Marcelo and Dunman (1997) reported that retinoic acid stimulates essential fatty acid-supplemented human keratinocytes. These results were observed in keratinocyte cultures grown in a serum-free medium (MCDB 153) that was supplemented with the protein growth factors, EGF, insulin, and BPE. Finally, Kamata et al. (1999) has reported the growth of oral keratinocytes in a novel protein-free defined medium call PF86-a (Rikimaru et al., 1990) with 85% serum-free medium, MCDB 153 (U.S. Pat. No. 4,673,649). No explanation or hypothesis was given as to what element(s) of the composition were responsible for the ability of these medium to support keratinocyte growth in the absence of protein growth.
A medium that eliminates the use of growth factors in a defined medium would have many technical and commercial benefits. In order to accomplish this goal, the inventor has replaced EGF and IGF-1 in a novel serum-free medium with retinyl acetate. Studies show that sustained growth of human keratinocytes is readily achieved in this improved serum-free medium. In addition, retinyl acetate stimulates proliferation at physiological concentrations unlike the reported effect of all-trans retinoic acid (Varani et al., 1989) and in HECK 109 serum-free medium supplemented with growth hormones (hydrocortisone, ethanolamine, and phosphoethanolamine).
Pellegrini in “Long Term Restoration of Damaged Corneal Surfaces with Autologous Cultured Corneal Epithelium”, Lancet, Vol. 349 (1997) discloses the culturing of corneal cells in Dulbecco, Vogt, Eagle's and Ham's F- 12 Media containing fetal bovine serum, insulin, transferin, EGF and cholera toxin. The authors' report that cells isolated from the central cornea (limbus) and bulbar conjunctiva could be grown in vitro and then transplanted to the human host.
U.S. Pat. No. 4,304,866 to Green et al. discloses an in vitro method for the formation of epithelial sheets from cultured keratinocytes. The Green method uses a serum containing medium and a feeder layer of murine (mouse) fibroblast cells to accomplish cell growth and differentiation. This procedure has serious limitations for large scale production of human epithelium as the use of serum inextricably confounds the culture of purely basal cells with the dynamics of serum-induced differentiation. The net result is that sub-cultivation of such cultures yields low (<5%) clonal efficiencies preventing step wise large scale build up of uncommitted pluripotent basal cells as a prelude to their conversion into usable sheets of transplantable, histologically-complete, human epithelium. Moreover, the process of Green et al. does not describe the formation of a histologically complete epidermis. The Green et al. procedure forms an epidermis lacking a stratum corneum which is necessary for maximizing the utility of the tissue.
Prior art methods have achieved a complete epidermis, but only in the presence of a complete skin starter sample and serum-containing media that are combined with an organotypic substratum containing growth factors produced by companion cells as disclosed in U.S. Pat. No. 4,485,096. The use of any organotypic substrate as well as feeder or companion cell types, e.g. fibroblasts, seriously limits the resulting products safety and economic viability. See Nanchahal, J. et al. in Lancet II(8656):191-193, (1989).
In order to remedy these deficiencies, the inventor has dispensed with serum-containing media, eliminated any substratum support, dispensed with the requirement for innumerable skin starter samples, and designed a novel and unobvious medium capable of supporting the growth and development of a complete epithelium. Moreover, the identification of essential process steps leading to a functional epithelium has been discovered and can be monitored with specific monoclonal antibodies. The prior art media which contain undefined serum and/or feeder cell factors and/or organotypic substrates and millimolar concentrations of Ca2+, high levels of buffers, inadequate levels of amino acids and incorrect osmolalities were not designed for the unlimited proliferation of undifferentiated basal cells. The prior art media allows cultures to spontaneously undergo maturation and uncontrolled differentiation. In contrast, the serum-free media described in this invention produces a complete epithelium.